Transient gene expression in CHO cells monitored with automated flow cytometry

Abstract

Transient gene expression is frequently used in industry to rapidly generate usable quantities of a protein from cultured cells. In gene therapy applications it is used to express a therapeutic protein in vivo. A quantitative assessment of the expression kinetics is important because it enables optimization and control of culture conditions for higher productivity. Previous experimental studies show a characteristic peak in average protein expression per cell after transfection followed by an exponential decrease of the expressed protein. Here, we show that the exponential decrease in single cell expression of enhanced Green Fluorescent Protein (eGfp) occurs in discrete steps. We attribute this to the absence of plasmid replication and to symmetric partitioning of plasmid and eGfp between dividing cells. This is reflected in the total eGfp in the bioreactor, which increased at a constant rate throughout the experiment. Additionally, the data provide a detailed time course of cell physiology during recovery from electroporation. The time course of cell physiology precisely indicates when the culture shifts growth phases. Furthermore, the data indicate two unique stationary phases. One type of stationary phase occurs when proliferation ceases while cells decrease their cell size, maintain granularity, and mean eGfp content decreases. The second type occurs when proliferation ceases while cells increase their cell size, increase granularity, and surprisingly maintain eGfp content. The collected data demonstrate the utility of automated flow cytometry for unique bioreactor monitoring and control capabilities in accordance with the US Food and Drug Administration's Process Analytical Technology initiative.